Physiological Responses, Fear-Related Behaviour And Meat Quality Of Broiler Chickens Subjected To Transportation And Other Stressors

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UNIVERSITI PUTRA MALAYSIA

PHYSIOLOGICAL RESPONSES, FEAR-RELATED BEHAVIOUR AND

MEAT QUALITY OF BROILER CHICKENS SUBJECTED TO

TRANSPORTATION AND OTHER STRESSORS

ABDULAZIZ AHMED AL-AQIL

FP 2009 15

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PHYSIOLOGICAL RESPONSES, FEAR-RELATED

BEHAVIOUR AND MEAT QUALITY OF BROILER CHICKENS

SUBJECTED TO TRANSPORTATION AND OTHER

STRESSORS

BY

ABDULAZIZ AHMED AL-AQIL

Thesis Submitted to the School of Graduate Studies, Universiti

Putra Malaysia in Fulfilment of Requirements for the Degree of

Doctor of Philosophy

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ii

DEDICATION

With appreciation and respect this thesis is dedicated to my beloved parents, my wife, sons, daughter, brothers and sisters.

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iii

Abstract of thesis presented to the Senate of Universisti Putra Malaysia in fulfillment of the requirement for the degree of Doctor of Philosophy

PHYSIOLOGICAL RESPONSES, FEAR-RELATED BEHAVIOUR AND MEAT QUALITY OF BROILER CHICKENS SUBJECTED TO

TRANSPORTATION AND OTHER STRESSORS

By

ABDULAZIZ AHMED AL-AQIL March 2009

Chairman: Professor Zulkifli Idrus, PhD Faculty: Agriculture

Five experiments were conducted to investigate the effects of pre-slaughter process and other stressors on adrenocortical (CORT) reaction, heterophil and lymphocyte ratios (HLR), fear-related behaviour (TI), meat quality, heat shock protein (hsp) 70 expression, performance, mortality and some blood parameters in broiler chickens.

In Experiment I, 200 day-old broiler chickens (Cobb x Cobb) showing short or long tonic immobility responses were classified as low-fear (STI) or high-fear (LTI) responders, respectively. On day 41, they were subjected to either crating or heat challenge (34+1oC) for 3 h and its effect on plasma corticosterone concentration, heterophil/lymphocyte ratios and heat shock protein 70 expression in brain tissue were determined. Crating and heat exposure elevated heterophil/lymphocyte ratios in both STI and LTI birds. Circulating corticosterone, however, was higher in LTI than STI birds following crating and heat challenge. Although differences between fear

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iv responder groups for hsp 70 were negligible prior to heat challenge, following 3 h of heat exposure, the response was greater for the LTI than the STI group. Both STI and LTI showed similar increases in hsp 70 following crating.

In Experiment II, 432 day-old broiler chicks (Cobb x Cobb) were housed either in an (i) environmentally controlled house (CH) which was maintained at 23±1 ºC from day 21 onwards or, (ii) conventional open-sided house (OH) with cyclic temperatures (minimum, 24 ºC; maximum, 34 ºC). Equal number of chicks of each housing system was subjected to either ad libitum feeding (AL) or 60% feed restriction on d 4, 5 and 6 (FR). On day 42, heterophil to lymphocyte ratios (HLR) were significantly lower in OH birds fed AL than those of CH. The CH birds had greater body weights, higher feed intake and better FCR than those of OH. Raising birds under OH, as measured by CORT, was more stressful than CH. The lower CORT in FR birds compared to their AL counterparts suggests improved heat tolerance in the former. Within the AL group, the OH chicks had lower HLR than CH and this could be associated with the more rapid growth rate in the latter. Raising birds in OH, where the birds were exposed to a wide variety of stimuli, shortened TI duration. The FR birds had shorter TI duration than their AL counterparts.

In Experiment III, two trials were conducted to investigate the effects of housing system and early age feed restriction on CORT, HLR, heat shock protein (hsp) 70 expression and some blood parameters in response to either day (11:00 h) or night (20:00 h) transportation. Chicks were raised either in an (i) environmentally controlled chamber (CH) which was maintained at 23±1 ºC from day 21, or (ii) conventional open-sided house (OH) with cyclic temperatures (minimum, 24 ºC;

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v maximum, 34 ºC). Equal number of chicks for each housing system was subjected to either ad libitum feeding (AL) or 60% feed restriction on d 4, 5 and 6(FR). On day 42, all the birds from each housing system-feeding regimen subgroup were subjected to road transport either at 11:00 h (Trial 1) or 20:00 h (Trial 2) for 6 hours (h) in open truck. Irrespective of time, transportation resulted in a marked elevation in HLR and CORT, The HLR and CORT data suggested that the magnitude of stress attributed to transportation increased with transit time. The chickens failed to habituate to stress following 6 h of transit. The OH chickens, as measured by HLR and CORT, were less distressed than their CH counterparts following crating and transportation. The improved ability of OH to cope with stress of transportation could be attributed to the greater hsp 70 response. Irrespective of time, FR dampened HLR reaction to transportation. For both day and night transportation, hsp 70 expression increased with transit time. Only day transportation had a consistent effect on serum levels of cholesterol, glucose and electrolytes. Chickens transported during the day had higher serum levels of sodium, chloride, glucose and cholesterol with transit time.

In Experiment IV, two trials were conducted to investigate the effects of housing system and early age feed restriction on measurements of TI duration, meat quality and some blood parameters. Chicks raised either in an (i) environmentally controlled chamber (CH) which was maintained at 23±1 ºC from day 21 old, or (ii) conventional open-sided house (OH) with cyclic temperatures (minimum, 24 ºC; maximum, 34 ºC). Equal number of chicks from each housing system was subjected to either ad libitum feeding (AL) or 60% feed restriction on d 4, 5 and 6(FR). On day 42, all the birds from each housing system-feeding regimen were subgrouped to road transport either at 11:00 h (Trial 1) or 20:00 h (Trial 2) for 6 h in an open truck.

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vi For both day and night transportation, TI durations increased with transit time. The OH birds which were exposed to a wider variety of stimuli, as measured by TI duration, were less fearful than their CH counterparts. Birds subjected to FR and transported during day had shorter TI duration than those fed AL. Irrespective of transportation time, the muscle glycogen content of broilers reduced markedly following transportation. Higher muscle glycogen content was noticed in the OH birds compared to CH following both day and night transportation. Muscle pH declined with transit time in birds transported during the day. Similar response was not noticed among birds transported at night. Following 2 h of transportation, OH birds had lower muscle pH but the reverse was observed after 6 h of transit. Both day and night transportation reduced serum lactic acid levels. Among the AL birds, the day time transportation lowered serum levels of lactic acid but converse was noted following night transportation. While serum creatine kinase (CK) activity was not affected by duration of transportation, birds transported at night showed lower serum levels of CK with transit time.

In Experiment V, the influence of pleasant and unpleasant experiences with human beings, and their combinations, on heat shock protein (hsp) 70 expression, and stress and fear responses in response to road transportation and disease resistance were studied in 750 day-old female broiler chicks. The pleasant treatment involved individual handling and gentle stroking for 30 sec daily from day 1 to 28 (PL). The unpleasant treatment involved individual handling, suspension by both legs, and exposure to noise (97 decibels) for 30 sec daily from day 1 to 28 (UNPL). The combination treatment involved exposure to either PL (day 1 to 14) and subsequently UNPL (day 15 to 28) or UNPL (day 1 to 14) and subsequently PL (day 15 to 28). On

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vii day 42, 60 birds per treatment were road transported for 3 h. Transportation resulted in higher HLR and CORT, and longer TI duration. The HLR of the PL birds was significantly lower than other groups. The CORT of PL and UNPL birds were not significantly different but lower than the other groups. Both PL-UNPL and UNPL-PL failed to attenuate HLR and CORT reactions following transportation. Although transportation had negligible effect on serum CK levels, the enzyme levels in PL birds were significantly lower than their control, PL-UNPL and UNPL counterparts. The PL and PL-UNPL birds were less fearful, as indicated by shorter TI duration. Irrespective of human treatment group, the amount of hsp 70 in the brain tissue was significantly higher following transportation. The hsp 70 reaction was significantly greater in the PL birds as compared to other groups. As measured by bursa to body weight ratio and bursal histological lesion score, human contact treatment had no consistent effect on resistance to infectious bursal disease.

It can be concluded that subjecting birds to pre-slaughter processes which include handling by humans, crating and transportation elicited both stress and fear reactions, and changes in meat quality and hsp 70 expression. Subjecting birds to early age feed restriction, pleasant human contact and raising birds in conventional open-sided housing system can improve the ability of birds to cope with environmental insults.

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viii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan ijazah Doktor Falsafah

REAKSI FISIOLOGI DAN KELAKUAN, DAN KUALITI

DAGING PADA AYAM YANG DISUBJEKKAN PADA PROSES

PRA-PENYEMBELIHAN DAN LAIN-LAIN TEKANAN

Oleh

ABDULAZIZ AHMED AL-AQIL

March 2009 Pengerusi: Professor Zulkifli Idrus, PhD Fakulti: Pertanian

Lima eksperimen dijalankan untuk mengkaji kesan proses pra penyembelihan dan lain-lain tekanan ke atas reaksi adrenokortikal (CORT), nisbah heterofil limfosit (HLR), tempoh tonic immobility (TI), kualiti daging, protein kejutan haba (hsp) 70, pencapaian, kadar kematian dan beberapa parameter darah dalam ayam pedaging. Dalam eksperimen I, 200 ekor anak ayam pedaging berumur sehari (Cobb x Cobb) yang menunjukkan reaksi tonic immobility pendek atau panjang masing-masing diklasifikasikan sebagai responden ketakutan rendah (STI) atau ketakutan tinggi ( LTI). Pada hari ke 41, ayam-ayam tersebut disubjekkan kepada kurungan dalam raga atau tegasan haba (34±1oC) selama 3 jam bagi menentukan kesan ke atas aras kortikosteron plasma, nisbah heterofil limfosit dan ekspresi hsp 70 dalam otak. Kurungan dalam raga dan pendedahan kepada tegasan haba meningkatkan nisbah heterofil / limfosit kedua-dua ayam STI dan LTI. Walau bagaimanapun, aras kortikosteron lebih tinggi pada ayam LTI berbanding STI selepas dikurung dan didedahkan kepada haba. Walaupun perbezaan antara kumpulan responden ketakutan untuk hsp 70 adalah kecil sebelum pendedahan pada kepanasan, kumpulan LTI

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ix menunjukkan reaksi lebih tinggi berbanding STI selepas 3 jam pendedahan. Kedua-dua STI dan LTI menunjukkan peningkatan yang sama dalam hsp 70 selepas dikurung.

Dalam eksperimen II, 432 ekor anak ayam berumur sehari (Cobb x Cobb) di tempatkan samada dalam (i) rumah persekitaran terkawal (CH) yang suhunya dikekalkan 23±1oC dari hari ke 21 dan seterusnya, atau (ii) rumah konvensional (OH) dengan suhu kitaran (minimum, 24oC; maximum,34oC). Anak ayam dari setiap sistem perumahan disubjekkan pada pemberian makanan secara ad libitum (AL) atau 60% sekatan makanan pada hari ke 4, 5 dan 6 (FR). Ayam CH mempunyai berat badan dan pengambilan makanan yang lebih tinggi, kadap pertukaran makanan yang lebih baik dari kumpulan OH. Memelihara ayam di bawah OH, seperti yang diukur dengan CORT, lebih tertekan berbanding CH. Nilai CORT yang lebih rendah dalam ayam FR berbanding ayam AL mencadangkan toleransi haba pada ayam FR. Dalam kumpulan AL, anak ayam OH mempunyai HLR yang lebih rendah berbanding CH dan ini mungkin dihubungkaitkan dengan kadar tumbesaran yang lebih cepat pada ayam CH. Memelihara anak ayam dalam OH, dimana ia terdedah kepada pelbagai ransangan memendekkan jangkamasa TI. Ayam FR mempunyai jangkamasa TI lebih pendek berbanding AL.

Dalam eksperimen III, dua ujian dijalankan untuk mengkaji kesan sistem perumahan dan kadar sekatan makanan awal hayat ke atas CORT, HLR, hsp 70 dan beberapa parameter darah pada ayam yang diangkut pada waktu siang (11:00 h) atau malam (20:00 h). Anak ayam dibesarkan samada dalam (i) rumah persekitaran terkawal (CH) di mana suhunya dikekalkan 23±1oC dari hari ke 21 dan seterusnya, atau (ii) rumah konvensional (OH) dengan suhu kitaran (minimum, 24oC; maksimum,34oC). Anak ayam disubjekkan pada pemberian makanan secara ad libitum (AL) atau 60%

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x sekatan makanan pada hari ke 4, 5 dan 6 (FR). Pada hari ke 42, semua ayam dari setiap subkumpulan sistem perumahan-kaedah pemberian makanan disubjekkan kepada pengangkutan darat samada pada 11:00 h (Ujian 1) atau 20:00 h ( Ujian 2) selama 6 jam dalam trak terbuka. Tanpa mengira masa pergerakan, pengangkutan menyebabkan peningkatan HLR dan CORT. Data HLR dan CORT menunjukkan secara tidak langsung bahawa magnitud tekanan disebabkan oleh pengangkutan meningkat dengan tempoh pengangkutan. Ayam gagal membiasakan diri pada tekanan selepas 6 jam pengangkutan. Ayam OH yang diukur melalui HLR dan CORT adalah kurang tertekan berbanding ayam CH yang setara dengannya selepas di kurung dalam raga dan diangkut. Peningkatan keupayaan OH untuk menghadapi tekanan pengangkutan mungkin disebabkan tindak balas hsp 70 yang lebih tinggi. Tanpa mengambil kira faktor masa, FR mengurangkan reaksi HLR pada pengangkutan. Untuk kedua-dua pengangkutan siang dan malam, ekspresi hsp 70 meningkat dengan masa pengangkutan . Hanya pergerakan siang mempunyai kesan konsisten ke atas paras serum kolesterol, glukos dan elektrolit. Ayam yang diangkut pada waktu siang mempunyai bacaan lebih tinggi pada paras serum natrium, klorida, glukos dan kolesterol dengan pergerakan masa.

Dalam eksperiment IV, dua ujian dijalankan untuk mengkaji tentang kesan sistem perumahan dan sekatan makanan awal hayat ke atas pengukuran jangkamasa TI, kualiti daging dan beberapa parameter darah. Anak ayam dibesarkan samada dalam (i) rumah persekitaran terkawal (CH) di mana suhunya dikekalkan 23±1oC dari hari ke 21 dan seterusnya, atau (ii) rumah konvensional (OH) dengan suhu kitaran (minimum, 24oC; maksimum,34oC). Anak ayam disubjekkan pada pemberian makanan secara ad libitum (AL) atau 60% sekatan makanan pada hari ke 4, 5 dan 6 (FR). Pada hari ke 42, semua ayam dari setiap subkumpulan sistem

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perumahan-xi kaedah pemberian makanan disubjekkan kepada pengangkutan darat samada pada 11:00 h (Ujian 1) atau 20:00 h ( Ujian 2) selama 6 jam dalam trak terbuka. Pada hari ke 42, semua ayam dari setiap subkumpulan sistem perumahan-kaedah pemberian makanan disubjekkan kepada pengangkutan darat samada pada 11:00 h (Ujian 1) atau 20:00 h ( Ujian 2) selama 6 jam dalam trak terbuka. Untuk kedua-dua pengangkutan siang dan malam, jangkamasa TI meningkat dengan tempoh pengangkutan. Ayam yang disubjekkan kepada FR dan pengangkutan siang hari mempunyai jangkama TI lebih pendek berbanding yang diberi pemakanan AL. Tanpa mengira masa pengangkutan, kandungan glikogen otot menurun dengan nyata selepas pengangkutan. Kandungan glikogen lebih tinggi pada ayam OH berbanding CH selepas kedua-dua pengangkutan siang dan malam. pH otot menurun dengan tempoh pengangkutan pada ayam yang diangkut pada waktu siang. Tindak balas yang serupa tidak didapati pada ayam yang diangkut pada waktu malam. Selepas 2 jam pengangkutan, ayam OH mempunyai pH otot lebih rendah tetapi keadaan sebaliknya selepas 6 jam pengangkutan. Kedua-dua pengangkutan siang dan malam menurunkan paras serum laktik. Di kalangan ayam AL, pengangkutan siang merendahkan paras serum laktik tetapi sebaliknya didapati selepas pengangkutan malam. Sementara itu, aktiviti serum kreatin kinase (CK) tidak dipengaruhi oleh jangkamasa pengangkutan. Ayam yang diangkut pada waktu malam menunjukkan paras serum CK lebih rendah dengan masa pergerakan.

Dalam eksperimen V, pengaruh pengalaman selesa atau tidak selesa dengan manusia ke atas ekspresi hsp 70, gerak balas takanan dan ketakutan akibat pengangkutan darat dan ketahanan penyakit diuji kepada 750 ekor anak ayam betina berumur sehari. Perlakuan selesa melibatkan memegang secara individu dan membelai selama 30 saat sehari dari hari pertama ke hari 28 (PL). Perlakuan tidak selesa melibatkan

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xii memegang secara individu, digantung dengan kedua belah kaki, dan didedahkan kepada kebisingan (97 desibel) selama 30 saat sehari dari hari pertama ke hari 28 (UNPL). Kombinasi perlakuan melibatkan pendedahan kepada samada PL (hari 1 hingga 14) dan diikuti UNPL (hari 15 hingga 28) atau UNPL (hari 1 hingga 14) dan diikuti PL (hari 15 hingga 28). Pada hari ke 42, 60 ekor ayam setiap perlakuan melalui proses pengangkutan darat selama 3 jam. Pengangkutan menyebabkan peningkatan HLR, CORT, dan jangkamasa TI lebih panjang. HLR ayam PL lebih rendah berbanding perlakuan lain. CORT bagi PL dan UNPL tidak banyak berbeza tetapi lebih rendah berbanding perlakuan lain. Kedua-dua PL- UNPL dan UNP-PL gagal mengurangkan reaksi HLR dan CORT selepas pengangkutan. Walaupun pengangkutan mempunyai kesan yang sangat kecil ke atas paras serum CK, paras enzim dalam ayam PL sangat rendah berbanding kumpulan PL-UNPL dan UNPL. Ayam PL dan PL-UNPL kurang takut seperti yang ditunjukkan oleh jangkamasa TI yang lebih pendek. Tanpa mengambil kira kumpulan perlakuan manusia, kuantiti hsp 70 dalam tisu otak sangat tinggi selepas pengangkutan. Reaksi hsp 70 sangat tinggi dalam ayam PL berbanding kumpulan lain. Seperti yang diukur oleh bursa kepada nisbah berat badan dan skor lesi histologi bursa, perlakuan sentuhan manusia tidak memberi kesan yang konsisten ke atas ketahanan kepada penyakit jangkitan bursa.

Sebagai kesimpulan, mendedahkan ayam pada proses pra-sembelih yang melibatkan tangkapan oleh manusia, dimasukan dalam sangkar dan pengangkutan mengakibatkan gerakbalas tegasan, ketakutan dan perubahan kauliti dagaing dan ekspressi hsp 70. Keadaan pemeliharaan dan kualiti kontak manusia member kesan besar ke atas gerakbalas ayam pada pengangkutan.

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ACKNOWLEDGEMENTS

The author would like to express his deep appreciation and authentic gratitude to esteemed Professor Dr. Zulkifli Idrus, chairman of the supervisory committee for his invaluable advice and guidance, fruitful criticisms, creditable suggestions, and patience in reading this dissertation throughout the labyrinth of the study.

The author would like to describe his utmost gratitude to the esteemed members of supervisory committee, Professor Dr. Mohamed Ali Rajion, Associate Professor Dr. Abdul Rahman Omar and Dr. Awis Qurni Sazili for their creditable guidance, constructive suggestions and critical reviewing of this dissertation.

The contributions of Professor Dr. Mohd. Hair-Bejo are highly appreciated. Deep appreciation and heartfelt gratitude are extended to Dr. Che Norma Mat Taib and to post graduate students from the Biologic Lab, Faculty of Veterinary Medicine.

The author gratefully acknowledges King Fisal University sponsorship, and heartfelt gratitude is extended to esteemed authorities from Universiti Putra Malaysia for supportive encouragement which enabled him to continue his studies here.

The author would like to extend the utmost gratitude with respect to the help from staffs at the Poultry Research Unit of Universiti Putra Malaysia, Mr. Ponnusamy Muniandy, Mr. Mazlan Hamzah and Mr. Hailunizam are gratefully acknowledged. The author would like to express his appreciation to Mr. Saparin Demin, Mr. Ibrahim Mohsin, Mr. Abdul Halin Isa, and Mr. Bakari Abd. Rahman (staffs from the Nutrition Lab, Animal Science Department, Faculty of Agriculture) and Mr. Saipuzaman Ali and Mr. Mohd. Halmi Othman (staffs from the Faculty of Veterinary Medicine) for their enthusiastic help and technical skills while conducting laboratory work.

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xiv Special thanks are due to all friends for their fine co-operations and moral support during the study and to all individuals. The author owed to those, who have made things run smoothly throughout the study.

Ultimately, the author is deeply indebted to his father, beloved mother, beloved wife, daughter, and sons for their endurance and precious supportive encouragement in various aspects during his study in Malaysia.

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xv

I certify that an Examination Committee met on 27 th

March 2009 to conduct the final examination of Abdulaziz Ahmed Al-Aqil on his Doctor of philosophy (Poultry Production) Thesis entitled “Physiology, fear-related behaviour and meat quality of broiler chickens subjected to transportation and other stressors” in accordance with Universiti Pertanian Malaysia (Higher act) 1980 and Universiti Pertanian Malaysia Regulations (Higher Degree) 1981. The committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follow:

ABD RAZAK ALIMON, PhD

Professor

Faculty of Agriculture Universiti Putra Malaysia (Chairman)

ENGKU AZAHAN ENGKU AHMAD, PhD

Associate Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia (Internal Examiner)

LOH TECK CHWEN, PhD

Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

WERNER BESSEI, PhD

Professor

Institute of animal husbandry and breeding University of Hohenheim

Garbenstr 17, Germany (External examiner)

_________________________________

HASANAH MOHD. GHAZALI, PhD

Professor/ Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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xvi This thesis submitted to the senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:

Zulkifli Idrus, PhD

Professor

Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Mohamed Ali B Rajion, PhD

Professor

Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Abdul Rahman Omar, PhD

Associate Professor

Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Awis Qurni Sazili, PhD

Senior Lecturer Faculty of Agriculture Universiti Putra Malaysia (Member)

________________________________ HASANAH MOHD GHAZALI, PhD Professor and Dean

School of Graduate Studies Universiti Putra Malaysia

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xvii

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declared that this thesis has not been previously or concurrently submitted for any other degree at UPM or any other institutions.

_____________________________ ABDULLAZIZ AHMED AL-AQIL Date:

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xviii TABLE OF CONTENTS page DEDICATION ii ABSTRACT iii ABSTRAK viii ACKNOWLEDGEMENTS xiii APPROVAL xv DECLARATION xvi

LIST OF TABLES xxii

LIST OF FIGURES xxv

LIST OF ABBREVIATIONS xxvi

CHAPTER

I GENERAL INTRODUCTION 1

II LITERATURE REVIEW 4

STRESS 4

The Concept of Stress 5

Environmental Stressors 6

Thermal Stress

PHYSIOLOGICAL RESPONSES TO STRESS

Glucocorticoids Hormones 8

Blood Cholesterol, Glucose and electrolytes Responses

to heat Stress 11

Early Age Stresses 13

Heat Shock Proteins 14

HANDLING AND TRANSPORTATION OF POULTRY

Handling and Crating 18

Transportation 20

FEAR RESPONSES

The Concept of Fear 21

Transportation and fear 24

Fear and Performance 25

Housing and Fear 26

Human-Animal Interaction and Fear 27

STRESS, FEAR AND MEAT QUALITY

Meat Quality 28

Post mortem changes 28

Post mortem pH decline 28

Post-mortem glycolysis 29

Factors influencing rate of pH and glycogen level 31

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xix

Fear and Meat Quality 33

III Crating and heat stress influences blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions

Introduction 34

Materials and Methods 37

Chickens and husbandry 37

Tonic immobility test 37

Crating 38

Heat challenge 38

Blood and brain samples 39

Determination of hsp 70 Concentration 39 Determination of Molecular Weight of hsp 70 41

Statistical analyses 46

Results 47

Tonic immobility duration 47

Effects of crating 47

Effects o f heat challenge 48

Discussion 50

Conclusion 52

IV Effects of the hot, humid tropical climate and early age feed restriction on stress, fear responses and performance in broiler chickens

Introduction 54

Chickens and husbandry 57

Growth performance 57

Blood sampling 58

Statistical analyses 59

Results 60

Discussion 66

Conclusion 71

V The effects of housing and early age feed restriction on broiler chickens subjected to road transportation. 1. Plasma

corticosterone concentration, heterophil to lymphocyte ratios and heat shock protein 70 expression

Introduction 72

General Methods 75

Trial 1 76

Road transportation 76

Blood samples 76

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xx Trial 2 77 Road transportation 77 Statistical analyses 78 Results 79 Discussion 95 Conclusion 101

VI The effects of housing and early age feed restriction on broiler chickens subjected to road transportation. 2. Fear reaction, meat quality and blood parameters

Introduction 102

General Methods 106

Trial 1 107

Tonic immobility tests 107

Blood samples 108

Meat quality determination 108

pH measurements 109 Trial 2 109 Road transportation 109 Statistical analyses 110 Results 111 Discussion 125 Conclusion 132

VII The combination of pleasant and unpleasant human contact affects stress and fear reactions, and disease resistance in broiler chickens

Introduction 133

Animals and housing 136

Treatment groups 136

Infectious Bursal Disease Challenge and Traits Measured 137

Tissue sample collection 138

Determination of bursa to body weight ratio 138 Determination of Heterophil/Lymphocyte Ratio 138 Lesion Scoring of Bursa of Fabricius 139

Histopathology 139

Blood parameters 141 Brain samples 141 Statistical analyses 142 Results 143 Discussion 153 Conclusion 160

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xxi

VIII GENERAL DISCUSSION AND CONCLUSION 161

REFERENCES 167

APPENDICES 200

BIODATA OF THE STUDENT 209

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xxii

LIST OF TABLES

Table Page

3.1 Mean (+SEM) heterophil / lymphocyte ratios (HLR) and heat shock protein (hsp) 70 densities by fear responder group and duration of

Crating 47

3.2 Mean (+SEM) plasma corticosterone concentrations (ng/ml) where fear responder group x duration of crating interactions were significant 48 3.3 Mean (+SEM) heterophil / lymphocyte ratios by fear responder group

and duration of heat exposure 49

3.4 Mean (+SEM) plasma corticosterone concentrations (ng/ml) and heat shock protein 70 densities where fear responder group x duration of heat exposure

interactions were significant 49

4.1 Mean (±SEM) body weights (BW), feed consumption (FC) and feed conversion ratios (FCR) in broiler chickens by housing system

and feeding regimen 61

4.2 The effect of housing system and feeding regimen in mortality

rate (%) in broiler chickens 62

4.3 Mean (±SEM) heterophil/lymphocyte ratio (HLR) in broiler chickens where feeding regimen by housing system interactions were significant 63 4.4 Mean (± SEM) The effect of housing system and feeding regimen

on plasma corticosterone concentration (ng/ml) and tonic immobility

duration (s)of broilers chickens 65

5.1 Mean (±SEM) heterophil/lymphocyte ratios (HLR) ), plasma corticosterone concentrations (ng/ml) and heat shock protein 70 density in broiler chickens where housing system duration of transportation interactions were

significant 81

5.2 Mean (±SEM) heterophil/lymphocyte ratios (HLR) in broiler chickens where feeding regimen by housing system interactions were significant 82

5.3 Mean (±SEM) plasma corticosterone concentrations (ng/ml) in broiler

chickens where feeding regimen by duration of transportation interactions

were significant 83

5.4 Mean (±SEM) heat shock protein 70 densities in broiler chickens where feeding regimen1 by housing system interactions were significant 84

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Table Page

5.5 Mean (± SEM) serum levels (mmol/L) of cholesterol, glucose, potassium and chloride by duration of transportation, housing system and feeding

regimen in broiler chickens 86

5.6 Mean (+SEM) serum levels of sodium (mmol/L) in broiler chickens where housing system by duration of transportation interactions were

significant 87

5.7 Mean (±SEM) heterophil/lymphocyte ratios (HLR), corticosterone

concentrations (ng/ml) and heat shock protein 70 densities where housing system by duration of transportation interactions were significant 90 5.8 Mean (+SEM) heat shock protein 70 densities in broiler chickens where

housing system by feeding regimen interactions were significant 91 5.9 Mean (± SEM) serum levels (mmol/L) of cholesterol, glucose, sodium

and chloride by duration of transportation, housing system and feeding

5.10 Mean (±SEM) serum levels of potassium (mmol/L) in broiler chickens where duration of transportation by housing system interactions were

significant 94

6.1 Mean (±SEM) tonic immobility (TI)durations (s) and number of attempts to induce TI by duration of transportation, housing system and feeding

6.2 Mean (±SEM) muscle glycogen contents (mg/g) in and serum level of creatine kinase (IU/L) in broiler chickens by duration of transportation, housing system and feeding regimen 114 6.3 Mean (+SEM) muscle pH values (unit) in broiler chickens where housing

system by duration of transportation interactions were significant 115 6.4 Mean (±SEM) serum levels of lactic acid (mmol/L) in broiler chickens

where feeding regimen by duration of transportation interactions were

significant 116

6.5 Mean (±SEM) serum levels of lactic acid (mmol/L) in broiler chickens where feeding regimen by housing system interactions were significant 117 6.6 Mean (±SEM) tonic immobility (TI) durations (s) by duration of

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6.7 Mean (±SEM) muscle glycogen contents (mg/g) in broiler chickens where housing system by duration of transportation interactions were

significant 120

6.8 Mean (+SEM) muscle pH (unit) values and serum levels of creatine kinase in broiler chickens by duration of transportation, housing system

and feeding regimen 122

6.9 Mean (±SEM) serum levels of lactic acid (mmol/L) in broiler chickens where feeding regimen by housing system interactions were significant 123 6.10 Mean (± SEM) serum levels of lactic acid (mmol/L) by duration of

transportation in broiler chickens 124 7.1 Mean (± SEM) heterophil/lymphocyte ratios (HLR) and heat shock

protein (hsp) 70 densities by duration of transportation and human

contact treatment in broiler chickens 144 7.2 Mean (± SEM) plasma corticosterone concentrations (ng/ml) by

duration of transportation and human contact treatment in broiler

Chickens 145

7.3 Mean (± SEM) tonic immobility (TI) durations (s) and number of attempts to induce TI by duration of transportation and human contact

treatment in broiler chickens 146

7.4 Mean (± SEM) bursa to body weight ratios (x10 -3) by human contact treatment and day post infection1 by IBDV in broiler

chickens 147

7.5 Mean (± SEM) heterophil counts (H), lymphocyte counts (L) and

heterophil/lymphocyte ratios (HLR) by human contact treatment and day post infection1 by IBDV in broiler chickens 148 7.6 Mean (± SEM) histological lesion scores1 of bursa tissue by human

contact treatment and day post infection2 by IBDV in broiler chickens 149

7.7 Mean (± SEM) serum levels of creatine kinase (IU/L) by duration of

transportation and human contact treatment in broiler chickens 151 7.8 Mean (± SEM) serum level of lactic acid, cholesterol, glucose,

total protein, sodium, potassium and chloride (mmol/L) by Duration of